Method of treating excessive fibrinolysis with synthetically modified trypsin inhibitors

ABSTRACT

Method of treating excessive fibrinolysis with a modified trypsin-callicrein inhibitor wherein the five carboxyl groups present in the unmodified material are amidated by 
     
         --(R)(R).sub.X W groups, 
    
     wherein R is prolyl or ##EQU1## X is an integer from 1 to 10, Y is hydrogen, alkyl, or substituted alkyl and W is --OH, --NH 2 , --NHC 2  H 5 , --OCH 3 , or --OC 2  H 5 .

This is a division of application Ser. No. 503,066 filed Sept. 4, 1974,now U.S. Pat. No. 3.953,417.

Many organs of mammals, especially the lung of bovines, contain atrypsin-callicrein inhibitor which can be isolated therefrom in variousways and which has been used now for 15 years with great success as apolyvalent proteinase and esterase inhibitor in the therapy of enzymedisorders.

This trypsin-callicrein inhibitor is a basic polypeptide with amolecular weight of 6500 and an isoelectric point of about 10.5. Itconsists of a single peptide chain of 58 aminoacids, the primarystructure of which is known from Biochemical and Biophysical ResearchCommunications Vol. 20, pages 463 to 468 (1965) and which iscross-linked by 3 disulfide bridges.

The high therapeutic value of this inhibitor is that it inhibits variousproteinases and esterases, for example trypsin, chymotrypsin, cathepsin,plasmin and callicrein. For this purpose, often considerable amounts ofthe inhibitor are injected, mainly intravenously. It has been found thatthe inhibitor is stored after a relatively short time, preferably in thekidneys of the test animal or of the human. It is assumed today thatthis is because the strongly basic inhibitor molecule is bound inunspecific manner to acid muco-polysaccharides or nucleic acids. Only1.5 % of the native inhibitor are excreted with the urine; in order tomake possible excretion of the preponderant residual quantity, theorganism first degrades an amino-acid each arginine or alanine) bound toan amino and carboxyl group, the inhibitor aggregates to a highermolecular form and is only then eluted very slowly from the kidney.

Tests with a chemically modified inhibitor in which a maleoyl group hadbeen introduced over all free amino groups and which proved to beinactive demonstrated that this substance, the isoelectric point ofwhich was considerably lower, was eluted essentially faster from thekidney and could be excreted with the urine. Thus, an inhibitor havingfull biological activity and a lower isoelectric point would haveconsiderable value. Now, in principle it is possible to reduce positivecharges by acylation of the free amino group and thus to lower theisoelectric point. However, all these reactions yield a reaction productwith reduced activity, if not even an inactive product, since the 5amino groups of the molecule must be assigned to 4 equivalent ε-aminogroups of the lysine and 1 α-amino group, while, on the other hand, thelysine in position 15 is decisive for the inhibitor action of thesubstance. Substitution of the amino group of lysine 15 causes the lossof the biological activity.

Now, it has been found that, unexpectedly, the activity of the inhibitoris only slightly modified or not at all by prolongation of the 5 presentcarboxyl groups with acid peptides, but that because of the lowering ofthe isoelectric point the modified inhibitor is excreted much fasterfrom the kidney than an unmodified molecule.

Thus, the present invention provides semi-synthetically preparedderivatives of the basic trypsin-callicrein inhibitor from organs ofmammals, in which the 5 carboxyl groups present in the peptide moleculeare amidated by peptide groups of the general formula I ##STR1## inwhich X represents an integer from 0 to 10, Y represents hydrogen or astraight chain or branched alkyl radical of 1 to 5 carbon atoms whichmay be substituted by a carboxyl, hydroxyl or carbonamide group, thegroup ##STR2## may also represent proline. It X = O, Y represents --CH₂COOH or --CH₂ --CH₂ --COOH, and if X is an integer from 1 to 10, atleast one lateral chain of Y is --CH₂ --COOH or --CH₂ --COOH or --CH₂--CH₂ --COOH. W represents --OH, but in the presence of at least twocarboxyl groups in the lateral chains, it may also represent --NH₂,NH--C₂ H₅, --OCH₃ or OC₂ H₅.

The invention furthermore relates to a process for preparing theabove-specified peptides, which comprises reacting thetrypsin-callicrein inhibitor, whose 5 primary amino groups are protectedby protective groups that can easily be split off with acids, withamino-acids or peptides of the general formula II ##STR3## in thepresence of 1-hydroxy-benzotriazole and dicyclohexylcarbodiimide andsubsequently splitting off the protective groups with the aid of acids.Y and X have the meanings given above, the carboxyl groups in Y,however, are present as tert.butyl esters. W' represents tert.butoxy,but if at least 2 tert.butyl ester groups are present in the lateralchains, then W' may also represent --NH₂, --NH--CH₃, --NH--C₂ H₅, --OCH₃or OC₂ H₅.

In order to be able to modify the trypsin-callicrein inhibitor at itscarboxyl groups, it is suitable to block the amino groups byamino-protective groups which can be easily split off with acids, forexample tert. alkoxy-carbonyl groups. The introduction of the often usedBoc-group can be carried out in various ways. Thus, the Boc-groups canbe introduced with Boc-azide as well as with Boc-active esters, forexample the p-nitrophenyl ester or the N-hydroxysuccinimide ester. Inall cases, a product is formed which is uniform in paperelectrophoresis. These N-protected compounds are reacted in the presenceof 1-hydroxy-benzotriazole and dicylohexylcarbodiimide with a compoundof the general formula II in dimethylformamide or dimethylacetamide. Theprotective groups are split off with trifluoroacetic acid or HCl/glacialacetic acid and the raw product is either dialyzed or chromatographedover Sephadex G 25.sup.(R), a cross-linked dextran gel. For furtherpurification, it is advisable also to chromatograph the dialyzed productlikewise over Sephadex G 25.sup.(R) or to subject it to partitionchromatography on Sephadex LH 20.sup.(R), a cross-linked alkylateddextran gel.

In order to lower the isoelectric point of the trypsin-callicreininhibitor, the amino-acid or peptide group to be condensed must containat least two carboxyl groups. Among the naturally occurring amino-acids,only aspartic acid and glutamic acid in their L- or D-form may be used.With dipeptides and higher peptides it is also possible to introduce, inaddition to the acid amino-acids, other aliphatic amino-acids, forexample glycine, alanine, leucine, valine, isoleucine, serine,threonine, asparagine, glutamine or proline in their L- or D-form. It ispreferred, however, with higher peptides also to use aspartic acid andglutamic acid as peptide components. If two or more carboxyl groups arepresent in the form of tert. butyl esters in the peptide to becondensed, further carboxyl groups in the form of amides or alkyl estersmay also be present. After acid treatment with trifluoroacetic acid orHCl/glacial acetic acid, there are obtained in this manner trypsininhibitors which, besides a low isoelectric point, additionally haveamide groups or alkyl ester groups.

In order to prove the decrease of the basic character and the increaseof the acid character in the inhibitor derivatives prepared according tothe invention, the native inhibitor and modified preparations weresubjected to micro-zone electrophoresis on cellulose-acetate foil in adiethyl-barbiturate buffer of pH 8.6.

The native inhibitor migrates in this case towards the cathode, whereasthe modified preparations migrate, depending on the modified group, moreor less far towards the anode. For the native inhibitor, the migrationdistance towards the cathode is about 3 mm. For the modifiedpreparation, in which the modified groups in the individual casescontain 1, 2, 3 or 4 acid amino-acids, the migration distances towardsthe anode are about 1.5 mm, 8 mm, 13.5 mm and 17 mm respectively.

Thus, the modification of the inhibitor leads to preparations the basiccharacter of which is reduced and the acid character of which isincreased. Modification with one group containing a single acidamino-acid already leads to such a lowering of the isoelectric pointthat at pH 8.6 the acid character slightly surpasses the basiccharacter.

The isoelectric points estimated from the dissociation constants of thefunctional groups of the inhibitor and its derivatives preparedaccording to the invention are about 10.5 for the native inhibitor andabout 8.1, 4.6, 4.35 and 4.1 for the modified preparations, the modifiedgroup containing one, two, three or four acid amino-acids.

For the fluorescence-microscopical proof of the storage of inhibitor andmodified inhibitor in the kidneys, 3 rabbits each having a weight of 2.5kg were injected intravenously at the same time with 20 mg of nativeinhibitor and 20mg of inhibitor modified according to the invention,respectively. After 2 days, the kidneys were removed, cut and incubatedwith a fluorescein-marked γ-globulin preparation which had been obtainedfrom the anti-serum of rabbits which had previously been vaccinatedseveral times with inhibitor and adjuvant according to Freund in orderto increase antibody formation.

The following picture was observed under the fluorescence-microscope:all kidney sections of the animals treated with the native inhibitorshowed great, partly confluing fluorescent spots distributed over thewhole visual field, whereas the sections of the kidneys of those animalsthat had obtained the modified inhibitor in most cases showed nofluorescence and contained small fluorescent spots only in a few cases.This test showed in distinct manner that the inhibitor with loweredisoelectric point is much less stored or not at all stored in thekidneys.

In preliminary tests by double diffusion, it had been confirmed that theantibody preparation used reacted not only towards the native inhibitorbut also towards the reaction products thereof obtained according to theinvention.

In order to determine the activity of the modified trypsin-callicreininhibitors, the following method was used:

The inhibitor solution was pre-incubated with a determined amount(corresponding to a determined activity of trypsin solution; after ashort time, a suitable trypsin substrate, for exampleN-benzoyl-arginine-4-nitroanilide (DAPA) was added and after some timeand after having stopped the reaction, the yellow coloration due top-nitro-aniline released by uninhibited trypsin was measuredquantitatively on a photometer at a suitable wave length, for example at405 mμ.

In particular, this process was carried out in that a quantity oftrypsin (trypsin, crystallized, analytically pure) which, as previouslydetermined, when combined within 30 minutes at 37° C with BAPA, causes acertain extinction at 405 mμ, was incubated with increasing amounts ofthe inhibitor in a constant volume. After a pre-incubation of 30minutes, the standard amount of BAPA was added. The reaction was stoppedafter 30 minutes at 37° C of BAPA incubation by the addition of diluteacetic acid and the yellow coloration was measured. When plotting in adiagram the inhibitor quantities added against the correspondingextinction values, a curve was obtained from which it was easy todetermine which quantity of inhibitor added was able to inhibit thetrypsin, which had always been added in constant amounts by one half.Since it was known which activity or what amount of trypsin had beenadded, it could be recalculated how many mg of inhibitor were able toinhibit how much trypsin.

In this test, about 1 mg of the modified inhibitor was found to be ableto inactivate about 3 mg of trypsin.

The trypsin -callicrein inhibitor modified according to the inventionserves as a medicament in the treatment of hemorrhages caused byexcessive fibrinolysis, for example in surgery in the case of prostatebleeding or disorders during the healing of wounds, in internal medicineas additional therapy in cases of hemophilia, in gynecology in cases ofplacenta praevia, fetal death in utero and atonic after-bleeding, andfor prophylaxis in the case of operations of parenchymatous organs, aswell as in cases of prostatectomies and fat embolisms.

The new medicaments of the invention are injected intravenously insterile isotonic solution or administered as a slow drip infusion afterdilution in infusion solutions, for example physiological salt solution.As doses, there may be administered 0.15 to 3 mg per kg.

The following Examples illustrate the invention.

EXAMPLE 1 Trypsin-callicrein inhibitor penta-L-glutamic acid

284 mg of DCC were added at 0° C to a suspension of 900 mg ofpenta-Boc-trypsin-callicrein inhibitor, 386 mg (1.3 mmoles) ofHCl.H-Glu(OBu^(t))-OBu^(t), 175 mg (1.3 mmoles) of HOBt and 0.17 ml (1.3mmoles) of N-ethylmorpholine in 10 ml of dimethylformamide and the wholewas stirred for 1 hour at 0° C and 24 hours at room temperature. Theprecipitate was filtered off with suction and the filtrate wasconcentrated. The residue was triturated with ether and dried. Yield:1.39 g.

The substance so obtained was dissolved in 10 ml of trifluoroaceticacid. The solution was allowed to stand for 30 minutes at roomtemperature and subsequently concentrated under reduced pressure. Theresidue was triturated with ether and filtered off with suction. Yield:1.17 g.

The substance so obtained was dialyzed against water and freeze-dried.Yield: 710.3 mg.

325 mg of the dialyzed and freeze-dried substance were chromatographedon Sephadex LH 20.sup.(R) (column 100 × 2.5 cm) in the system glacialacetic acid/butanol/water 42:20:228 mg of a pure fraction were isolated.

Amino-acid analysis: ratio Gly:Glu = 6:8.5 (theory 6:8).

EXAMPLE 2 Trypsin-callicrein inhibitor penta-(Glu-Glu-OH)

900 mg of penta-Boc-trypsin-callicrein inhibitor were reacted with 625mg (1.3 mmoles) of HCl.H-Glu(OBu^(t))-Glu(OBu^(t))-OBu^(t) in a manneranalogous to that described in Example 1. After treatment withtrifluoroacetic acid and subsequent dialysis, 770.4 mg of raw substancewere obtained. 500 mg of this raw substance were purified as describedin Example 1.

Yield: 382 mg of a pure fraction.

Amino-acid analysis: ratio Gly:Glu = 6:14.6 (theory 6:13).

EXAMPLE 3 Trypsin-callicrein inhibitor penta-(Glu-Glu-Glu-OH)

900 mg of penta-Boc-trypsin-callicrein inhibitor were reacted with 867mg (1.3 mmoles) of HCl.H-Glu(OBu^(t))-Glu(OBu^(t))-Glu(OBu^(t))-OBu^(t)in a manner analogous to that described in Example 1. After treatmentwith trifluoroacetic acid, 1.355 g of raw substance were obtained whichwere chromatographed in 0.1 m acetic acid over Sephadex G 25 .sup.(R)(column 200 × 4 cm).

Yield: 674.9 mg of a pure fraction.

Amino-acid analysis: ratio Gly:Glu = 6:18.3 (theory 6:18).

EXAMPLE 4 Trypsin-callicrein inhibitor penta-(Glu-Glu-Glu-Glu-OH)

900 mg of penta-Boc-trypsin-callicrein inhibitor were reacted with 1.11g (1.3 mmoles) ofHCl.H-Glu(OBu^(t))-Glu(OBu^(t))-GLu(OBu^(t))-Glu(OBu^(t))-OBu.sup.t in amanner analogous to that described in Example 1. After treatment withtrifluoroacetic acid, 1.15 g of raw substance were obtained which werechromatographed in 0.1 m acetic acid over Sephadex G 25 .sup.(R) (column200 × 4 cm).

Yield: 745 mg of a pure fraction.

Amino-acid analysis: ratio Gly:Glu = 6:23.4 (theory 6:23).

EXAMPLE 5 Trypsin-callicrein inhibitor penta-aspartic acid

900 mg of penta-Boc-trypsin-callicrein inhibitor were reacted with 365mg (1.3 mmoles) of HCl.H-Asp(OBu^(t))-OBu^(t) in a manner analogous tothat described in Example 1. After treatment with trifluoroacetic acid,1.083 g of raw substance were obtained which were chromatographed in 0.1macetic acid over Sephadex G 25 .sup.(R) (column 200 × 4 cm).

Yield: 707.6 mg of a pure fraction.

Amino-acid analysis: ratio Gly-Glu-Asp = 6:3.2:10.5 (theory 6:3:10).

EXAMPLE 6 Trypsin-callicrein inhibitor penta-(Glu-Asp-OH)

900 mg of penta-Boc-trypsin-callicrein inhibitor were reacted with 610mg (1.3 mmoles) of HCl.H-Glu(OBu^(t))-Asp(OBu^(t))-OBu^(t) in a manneranalogous to that described in Example 1. After treatment withtrifluoroacetic acid, 1.1 g of raw substance were obtained which werechromatographed in 0.1 m acetic acid over Sephadex G 25 .sup.(R) (column200 × 4 cm).

Yield: 759.3 mg of a pure fraction.

Amino-acid analysis: ratio Gly:Glu:Asp = 6.0:8.5:10.5 (theory 6:8:10).

EXAMPLE 7 Trypsin-callicrein inhibitor penta-Glu-Glu-Asp)

900 mg of penta-Boc-trypsin-callicrein inhibitor were reacted with 848mg (1.3 mmoles) ofHCl.H-Glu(OBu^(t))-Glu(OBu^(t))-Glu(OBu^(t))-Asp(OBu^(t))-OBu.sup.t in amanner analogous to that described in Example 1. After treatment withtrifluoracetic acid, 1,266 g of raw substance were obtained which werechromatographed in 0.1 m acetic acid over Sephadex G 25 .sup.(R) (column200 × 4 cm).

Yield: 727 mg of a pure fraction.

Amino-acid analysis: ratio Gly:Gly:Asp = 5.5:13.6:10.0 (theory 6:13:10).

EXAMPLE 8 Trypsin-callicrein inhibitor penta-(Asp-Glu-Asp)

900 mg of penta-Boc-trypsin-callicrein inhibitor were reacted with 830mg (1.3 mmoles) of HCl.H-Asp(OBu^(t))-Glu(OBu^(t))-Asp(OBu^(t))-OBu^(t)in a manner analogous to that described in Example 1. After treatmentwith trifluoroacetic acid, 1.25 g of raw substance were obtained whichwere chromatographed in 0.1 m acetic acid over Sephadex G 25.sup.(R)(column 200 × 4 cm).

Yield: 628.4 mg of a pure fraction.

Amino-acid analysis: ratio Gly:Glu:Asp = 6.0:8.0:15:1 (theory 6:8:15).

EXAMPLE 9 Trypsin-callicrein inhibitor pental(Glu-Asp-Glu-Asp)

900 mg of penta-Boc-trypsin-callicrein inhibitor were reacted with 1.07g (1.3 mmoles) ofHCl.H-Glu(Obu^(t))-Asp(OBu^(t))-Glu(OBu^(t))-Asp(OBu^(t))-OBu.sup.t in amanner analogous to that described in Example 1. After treatment withtrifluoroacetic acid, 1.54 g of raw substance were obtained which werechromatographed in 0.1 m acetic acid over Sephadex G 25.sup.(R) (column200 × 4 cm).

Yield: 634 mg of a pure fraction.

Amino-acid analysis: ratio Gly:Glu:Asp = 5.7:13.0:15.6 (theory 6:13:15).

EXAMPLE 10 Trypsin-callicrein inhibitor penta-(Glu-D-Glu-OMe)

900 mg of penta-Boc-trypsin-callicrein inhibitor were reacted with 597mg (1.3 mmoles) of HCl.H-Glu(OBu^(t))-D-Glu(OBu^(t))-OMe (J. Chem. Soc.Perkin I 1972, page 1) in a manner analogous to that described inExample 1. After treatment with trifluoroacetic acid, 1.2 g of rawsubstance was obtained which was chromatographed in 0.1 m acetic acidover Sephadex G 25.sup.(R) (column 200 × 4 cm).

Yield: 654 mg of a pure fraction.

Amino-acid analysis: ratio Gly:Glu = 6:13.7 (theory 6:13).

EXAMPLE 11 Trypsin-callicrein inhibitor penta(Asp-Glu-NH₂)

900 mg of penta-Boc-trypsin-callicrein inhibitor were reacted with 533mg (1.3 mmoles) of HCL.H-Asp(OBu^(t))-Glu(OBu^(t))-NH₂ (obtained bycatalytic hydrogenation of Z-Asp(OBu^(t))-Glu(OBu^(t))-NH₂, whichisdescribed in J. Chem. Soc. C (1968), page 531, in a manner analogous tothat described in Example 1. After treatment with trifluoracetic acid,1.18 g of raw substance was obtained, which was chromatographed in 0.1 macetic acid over Sephadex G 25 .sup.(R) (column 200 × 4 cm).

Yield: 640 mg of a pure fraction.

Amino-acid analysis: ratio Gly:Glu:Asp = 6.0:8.6:10.3 (theory 6:8:10).

EXAMPLE 12 Trypsin-callicrein inhibitor penta(Asp-Ser-Asp-OH)

900 mg of penta-Boc-trypsin-callicrein inhibitor were reacted with 702mg (1.3 mmoles) of HCl.H-Asp(OBu^(t))-Ser-Asp(OBu^(t)) (J. Chem. Soc. C.Org., 1969, page 2218) in a manner analogous to that described inExample 1. After treatment with trifluoroacetic acid, 1.3 g of rawsubstance were obtained which were chromatographed in 0.1 m acetic acidover Sephadex G 25.sup.(R) (column 200 × 4 cm).

Yield: 710 mg of a pure fraction.

Amino-acid analysis: ratio (Gly:Asp:Ser = 6:15.5:4.9 (theory 6:15:6).

EXAMPLE 13 Trypsin-callicrein inhibitor penta(Glu-Glu-D-Glu-OH)

900 mg of penta-Boc-trypsin-callicrein inhibitor were reacted with 867mg (1.3 mmoles) ofHCL.H-Glu(OBu^(t))-Glu(OBu^(t))-D-Glu(OBu^(t))-OBu^(t) in amanneranalogous to that described in Example 1. After treatment withtrifluoroacetic acid, 1.25 g of raw substance were obtained which werechromatographed over Sephadex G 25.sup.(R) (column 200 × 4 cm).

Yield: 683.2 mg. of a pure fraction.

Amino-acid analysis: ratio Gly:Glu = 6:00:18.07 (theory 6.18).

EXAMPLE 14 Trypsin-callicrein inhibitor penta(D-Gly-D-Glu-D-Glu-OH)

900 mg of penta-Boc-trypsin-callicrein inhibitor were reacted with 867mg (1.3 mmoles) ofHCl.H-D-Glu(OBu^(t))-D-Glu(OBu^(t))-D-Glu(OBu^(t))-OBu^(t) in a manneranalogous to that described inn Example 1. After treatment withtrifluoroacetic acid, 1.52 g of raw substance were obtaind which werechromatographed in 0.1 m acetic acid over Sephadex G 25.sup.(R) (column200 × 4 cm).

Yield: 652.9 mg of a pure fraction.

Amino-acid analysis: ratio Gly:Glu = 6:18.88 (theory 6:18).

Preparation of the Starting Substances a.Z-Glu(OBu^(t))-Glu(OBu^(t))-OBu^(t)

10.4 g (20 mmoles) of Z-Glu(OBu^(t))-OH.DCHA were stirred into 100 ml ofether and 20 ml of IN H₂ SO₄ at 0° C. The ether phase wasdried withsodium sulfate and concentrated. The residue was dissolved in 50ml ofdimethylformamide. This solution was combined with 5.91 g (20 mmoles)ofHCl.H-Glu(OBu^(t))-OBu^(t), 2.7 g (20 mmoles) of HOBt, 2.6 ml ofN-ethylmorpholine and, at 0° C, 4.4 g of DCC (as a solution indimethylformamide). The whole was stirred for 1 hour 0° C, for 2 hoursat room temperature and allowed to stand overnight at room temperature.The next day, the precipitate was filtered off with suction and thefiltrate was concentrated. The residue was dissolved in ethyl acetateand the solution was washed successively with NaHCO₃ solution, KHSO₄solution, NaHCO₃ solution and water, dried over sodium sulfate andconcentrated. The residue was chromatographed in ethyl acetate overabout 30 g of basic Al₂ O₃ (Woelm, activity degree I). The eluate wasconcentrated and dried in a high vacuum. Yield: 10 g ofoil (86 %). Aftersome time, the oil crystallized; melting point 88°C.

C₃₀ H₄₆ N₂ O₉ (578.7)--Calc.: C 62.26, H 8.01, N, 4.84.Found: C 62.3, H8.1, N, 5.1.

b. HCl.H-Glu(OBu^(t))-Glu(OBu^(t))-OBu^(t)

9.6 g of Z-Glu(OBu^(t))-Glu(OBu^(t))-OBu^(t) were dissolved in methanol,combined with a Pd catalyst and hydrogenated with the aid of anautotitrator with addition of 2N-methanolic HCl at pH 4.5. When thehydrogenation was completed, the catalyst was filtered off with suctionand the filtrate was concentrated.

Yield: 7.3 g of oil (91 %).

C₂₂ H₄₀ N₇.HCl (481.04)

c. Z-Glu(OBu^(t))-Glu(OBu^(t))-Glu(OBu^(t))-OBu^(t)

6.65 g (12.9 mmoles) of Z-Glu(OBu^(t))-OTcp were added to a solution of6.2 g (12.9 mmoles) of HCl.H-Glu(OBu^(t))-Glu(OBu^(t))-OBu^(t), 1.74g(12.9 mmoles of HOBt, 1.68 ml (12 mmoles) of N-ethylmorpholine in 60 mlof dimethylformamide, the whole was stirred for 5 minutes andconcentratedin a high vacuum. The residue was dissolved in ethyl acetateand treated asdescribed under (a). After drying over sodium sulfate, thesolution was concentrated and the residue was triturated with petroleumether. The whole was cooled to 0° C and filtered with suction. Yield:6.3 g (70 %). Melting point: 111° to 113° C.

[α]²² _(D) = -29.8° (c = 1, methanol)

C₃₉ H₆₁ N₃ O₁₂ (763.94) Calc.: C 61.35, H 8.04, N 5.51. Found: C 61.2, H8.0, N 5.6.

d. HCl.H-Glu(OBu^(t))-Glu(OBu^(t))-Glu(OBu^(t))-OBu^(t)

5.8 g (7.6 mmoles) of Z-Glu(OBu^(t))-Glu(OBu^(t))-Glu(OBu^(t))-OBu^(t)were hydrogenatedin methanol as described under b). Yield: 4.8 g of oil(95 %).

C₃₁ H₅₅ N₃ O₁₀ . HCl (666.3)

e. Z-Glu(OBu^(t))-Glu)OBu^(t)).Glu(OBu^(t))-Glu(OBu^(t) -OBu^(t)

3.05 g (5.85 mmoles) of Z-Glu(OBu^(t))-OTcp were added to a solution of3.9 g (5.85 mmoles) ofHCl.H-Glu(OBu^(t))-Glu(OBu^(t))-Glu(OBu^(t))-OBu^(t), 0.79 g(5.85mmoles) of HOBt and 0.76 ml (5.85 mmoles) of N-ethylmorpholine in50 ml of dimethylformamide. The whole was stirred for 1 hour at roomtemperature and worked up as described under (c). Yield: 4.75 g (86 %).Melting point:119° to 120° C.

[α]_(D) ²² = -29.1° (c = 1, methanol)

C₄₈ H₇₆ N₄ O₁₅ (949.2)--Calc.: C 60.75, H 8.08, N 5.91.Found: C 60.6, H8.0, N 5.8.

f. HCl.H-Glu(OBu^(t))-Glu(OBu^(t))-Glu(OBu^(t))-Glu(OBu^(t))-OBu.sup.t

4.4 g (4,63 mmoles) ofZ-Glu(OBu^(t))-Glu(OBu^(t))-Glu(OBu^(t))-Glu(OBu^(t))-OBu^(t) werehydrogenated catalytically in methanol as described under b). Yield:3.85 g of oil (98 %).

C₄₀ H₇₀ N₄ O₁₃. HCl (851.5)

g. Z-Glu(OBu^(t))-Asp(OBu^(t))-OBu^(t)

52 g (0.1 mole) of Z-Glu(OBu^(t))-OH.DCHA were stirred at 0° C in etherand 100 ml of lN-H₂ SO₄. The ether phase was washed once with water,dried over sodium sulfate and concentrated. The residue was dissolved in200 ml of dimethylformamide. This solution was combined with 28.1 g (0.1mole) of HCl.H-Asp(OBu^(t))-OBu^(t), 13.5 g of HOBt (0.1 mole), 13 ml ofN-ethylmorpholine (0.1 mole) and, at 0° C, with 22 g of DCC (as solutionin dimethylformamide). The process was then carried out as describedunder (a). The residue crystallized upon trituration withpetroleumether. Yield: 42.9 g (76 %); melting point: 128 to 130° C.For furtherpurification, the substance was dissolved in ether and chromatographedover 120 g of basic Al₂ O₃. Yield: 34.7 g; melting point 131° to 132° C.

[α]_(D) ²² = -17. 9° (c = 1, methanol)

C₂₉ H₄₄ N₂ O₉ (564.7)-- Calc.: C 61.70, H 7.86, N 4.96.Found: C 61.7, H7.8, N 4.9.

h. HCl.H-Glu(OBu^(t))-Asp(OBu^(t))-OBu^(t)

34.5 g (62 mmoles) of Z-Glu(OBu^(t))-Asp(OBu^(t))-OBu^(t) werehydrogenated catalytically in methanol as described under (b). Yield:29.3g of oil (100 %).

C₂₁ H₃₈ N₂ O₇ . HCl (467.01)

i. Z-Glu(OBu^(t))-Glu(OBu^(t))-Asp(OBu^(t))-OBu^(t)

13.82 g (26.7 mmoles) of Z-Glu(OBu^(t))-OTcp were added at roomtemperature to a solution of 12.5 g (26.7 mmoles) ofHCl.H-Glu(OBu^(t))-Asp(OBu^(t))-OBu^(t), 3.6 g (26.7 mmoles) of HOBt,3.5 ml (26.7 mmoles)) of N-ethylmorpholine in 100 ml ofdimethylformamide. The whole was allowed to stand for 1 hour and workedupas described under (c). Yield: 27.4 g of oil. For furtherpurification, theoil was dissolved in ether and chromatographed over 75g of basic Al₂ O₃.

Yield: 17.65 g of amorphous mass (88 %).

[α]_(D) ²² = -23.9° (c = 1, methanol).

C₃₈ H₅₉ N₃ O₁₂ (749.9)-- Calc.: C 60.86, H 7.93, N 5.61. Found: C 60.3,H 7.9, N 5.6.

j. HCl.H-Glu(OBu^(t))-Glu(OBu^(t))-Asp(OBu^(t))-OBu^(t)

17 g (22.7 mmoles) of Z-Glu(OBu^(t))-Glu(OBu^(t))-Asp(OBu^(t))-OBu^(t)were hydrogenatedcatalytically in methanol in a manner analogous to thatdescribed under (b).

Yield: 14.34 g of amorphous mass (97 %)

C₃₀ H₅₃ N₃ O₁₀ . HCl (652.2)

k. Z-Asp(OBu^(t))-Glu(OBu^(t))-Asp(OBu^(t))-OBu^(t)

11.25 ml (26.7 mmoles) of Z-Asp(OBu^(t))-ONSu were added to a solutionof12.5 g (26.7 mmoles) of HCl.H-Glu(OBu^(t))-Asp(OBu^(t) - OBu^(t) and3.5 ml (26.7 mmoles) of N-ethylmorpholine in 100 ml of dimethylformamideand the whole was allowed to react for about 20 hours at roomtemperature.Working up was effected as described under (c). Forpurification, the residue was dissolved in ether and chromatographedover 50 g of basic Al₂ O₃.

Yield: 13.4 g of amorphous substance (68 %).

[α]_(D) ²² = -23.9° (c = 1, methanol)

C₃₇ H₅₇ N₃ O₁₂ (735.9)-- Calc.: C 60.4, H 7.81, N 5.71.Found: C 60.0, H7.1, N 5.6.

l. HCl.H-Asp(Obu^(t) -Glu(OBu-^(t))-Asp(OBu^(t))-OBu^(t)

12.8 g (17.4 mmoles) of Z-Asp(OBu^(t))-Glu(OBu^(t))-Asp(OBu^(t))-OBu^(t)were hydrogenatedcalalytically in methanol in a manner analogous to thatdescribed under (b).

Yield: 10.4 of amorphous substance (94 %).

C₂₉ H₅₁ N₃ O₁₀ . HCl (638.2)

m. Z-Glu(OBu^(t))-Asp(OBu^(t))-Glu(OBu^(t))-Asp(OBU^(t))-OBu^(t)

5 g (10 mmoles) of Z-Glu(OBu^(t))-OTcp were added to a solution of 6.4 g(10 mmoles) of HCl.H-Asp(OBu^(t))-Glu(OBu^(t))-Asp(OBu^(t))-OBu^(t),1.35 g (10 mmoles) of HOBt and 1.3 ml (10 mmoles) of N-ethylmorpholinein 40 ml of dimethylformamide, the whole was stirred for 1 hour at roomtemperature and worked up in a manner analogous to that described underc). The residue was triturated with petroleum ether. Yield: 7.4 g (80%); melting point 196° C. [α]_(D) ²² = -24.6° (c = 1, methanol) C₄₆ H₇₂N₄ O₁₅ (921.1)-- Calc.: C 59.98, H 7.88, N 6.08. Found: C 59.6, H 7.8, N6.1.

n. HCl.H-Glu(OBu^(t))-Asp(OBU^(t))-Glu(OBu^(t))-Asp(OBU^(t))-OBu.sup.t

7 g (7.6 mmoles) ofZ-Glu(OBu^(t))-Asp(OBu^(t))-Glu(OBu^(t))-Asp(OBu^(t))-OBu^(t) werehydrogenated catalytically in methanol as described under (b).

Yield: 6.1 g of amorphous substance (97.5 %) C₃₈ H₆₆ N₄ O₁₃ . HCl(823.4)

o. Penta-Boc-trypsin-callicrein inhibitor

(α ) 1.3 g of trysin-callicrein inhibitor were dissolved in 10 ml ofwater. The solution was combined with 50 ml of dimethylformamide and 4.4ml of saturated NaHCO₃ solution. Then, 430 mg of Boc-ONSu were added,thewhole was stirred for 1 hour at room temperature and allowed to standovernight. The next day, it was acidified to pH 5 with 2N-acetic acid.Theclear solution was concentrated in a high vacuum. The residue wastritured with water, filtered off suction, well washed with water anddried. It wasagain triturated with ethyl acetate, filtered off withsuction and dried. Yield 880 mg.

Paper electrophoresis showed, in an acid medium, a uniform substancewhich was different from the trypsin-callicrein inhibitor.

(β) 1.3 g of trypsin-callicrein inhibitor were suspended in 20 ml ofdi-methylformamide. This suspension was combined with 270 mg of HOBt,480 mg of Boc-ONp and 0.14 ml of 1,1',4,4'-tetramethylguanidine. Further0.7 ml of 1,1'-4,4'-tetramethylguanidine and 480 mg of Boc-ONp wereadded in several portions. The whole was stirred for a total of 2 daysat room temperature, concentrated and the residue was stirred with ethylacetate. The precipitate was triturated with methanol and filtered offwith suction. Yield: 950 mg. The product so obtained was found to beidentical in paper electrophoresis with the product obtained accordingto (α).

γ. 7 g of trypsin-callicrein inhibitor were dissolved in 77 ml of water.315 ml of dimethylformamide, 24 ml of saturated NaHCO₃ solution and 14ml of Boc-azide were added. The whole was stirred for 5 hours at 35° C,acidified with 2N-acetic acid to pH 5 and concentrated in a high vacuum.The residue was triturated with water and filtered off with suction.Yield: 6.5 g. The product obtained was found tobe identical in paperelectrophoresis with the substance obtained accordingto (α).

p. HCl.H-D-Glu(OBu^(t))-D-Glu(OBu^(t))-D-Glu(OBt^(t))-OBu^(t)

The compound was prepared according to the methods described under (a),(b), (c) and (d) from the corrresponding D-glutamic acid derivatives.The melting points corresponded to those of the L-compounds. Thespecific rotations of the D-compounds were also found to correspond intheir valuesto those of the L-compounds, but had a reversed sign.

q. Z-Glu(OBu^(t))-D-Glu(OBu^(t))-OBu^(t)

5.17 g of Z-Glu(OBu^(t))-OTcp and 2.6 ml of N-ethylmorpholine were addedto a solution of 2.96 g (10 mmoles) of HCl.H-D-Glu(OBu^(t))-OBu^(t) and1.35 g of HOBt in 20 ml of dimethylformamide. After 1 hour, the wholewas concentrated, the residue was dissolved in ethyl acetate and washedwith water, saturated NaHCO₃ solution, KHSO₄ solution and NaHCO₃solution, dried over Na₂ SO₄ and concentrated. For purification, it waschromatographed in methylene chloride over 70 g of silica gel. Afterelution with 400 ml of methylene chloride, the substancewas eluted withthe methylene chloride/acetone mixture (9:1). Yield: 5.3 g of oil.

n. HCl.H-Glu(OBu^(t))-D-Glu(OBu^(t))-OBu^(t)

5.3 g of oily Z-Glu(OBu^(t))-D-Glu(OBu^(t))-OBu^(t) werehydrogenatedcatalytically in a manner analogous to that of Example (b).

Yield: 3.64 g of oil.

s. Z-Glu(OBu^(t))-Glu(OBu^(t))-D-Glu(OBu^(t))-OBu^(t)

0.97 ml of N-ethylmorpholine and 3.88 g of Z-Glu(OBu^(t))OTcp were addedto a solution of 3.6 g of HCl.H-Glu(OBu^(t))-D-Glu(OBu^(t))-OBu^(t) and1.01 g of HOBt in 20 ml of dimethylformamide. After 1 hour, the productwas worked up in a manner analogous to that of Example (c).

Yield: 4.35 g; melting point 129° to 130° C. [α]_(D) ²² = -10.4° (c = 1,in methanol)

t. HCl.H-Glu(OBu^(t))-Glu(OBu^(t))-D-Glu(OBu^(t))-OBu^(t)

4.13 g of Z-Glu(OBu^(t))-Glu(OBu^(t))-D-Glu(OBu^(t))-OBu^(t) werehydrogenated catalytically in a manner analogous to that of Example (b).Yield: 3.3 g of amorphous substance.

    ______________________________________                                        Abbreviations                                                                 ______________________________________                                        Boc           tert.-Butyloxycarbonyl                                          Z             Benzyloxycarbonyl                                               OBu.sup.t     tert.-Butyl ester                                               ONp           4-Nitrophenyl ester                                             ONSu          N-Hydroxysuccinimide ester                                      OTcp          2,4,5-Trichlorophenyl ester                                     DCHA          Dicyclohexylamine                                               DCC           Dicyclohexyl-carbodiimide                                       HOBt          1-Hydroxybenzotriazole                                          ______________________________________                                    

We claim:
 1. A method for treating excessive fibrinolysis caused by disfunctions of proteinases and esterases in a patient suffering therefrom, which method comprises administering to said patient by intravenous injection or infusion an effective amount of a modified trypsin-callicrein inhibitor wherein the five carboxyl groups present in the unmodified material are amidated by peptide groups of the formula

    --R(R).sub.X W,

wherein R is ##EQU2## or prolyl; Y is hydrogen, alkyl having 1 to 5 carbon atoms, or alkyl having 1 to 5 carbon atoms substituted by a carboxyl, hydroxyl, or carbonamide group; X is an integer from 0 to 10 such that, if X = 0, then R is ##EQU3## and, if X is an integer from 1 to 10, then at least one R is ##EQU4## and W is --OH, --NH₂, --NHC₂ H₅, --OCH₃, or --OC₂ H₅ when --R(R)_(X) -- contains at least two ##EQU5## wherein Y contains carboxy, but is otherwise --OH, said modified inhibitor being administered in combination with a physiologically tolerated solvent therefor.
 2. A method as in claim 1 wherein from 0.15 to 8 mg of said modified inhibitor are administered per kg of body weight. 